Continuous subcutaneous injection reduces polymorphonuclear leukocyte activation by granulocyte colony-stimulating factor

The use of granulocyte colony stimulating factor (G-CSF) for recovery from neutropenia has been established; however, acute lung injury due to G-CSF-induced polymorphonuclear leukocyte (PMN) activation is a serious complication. This study was designed to compare the activation of PMN with single bolus administration and continuous administration of G-CSF. Healthy volunteers (age 33.8 +/- 1.4 yr; n = 6) received a single bolus injection of 50 microm/m2 of G-CSF (SI; n = 6) or continuous subcutaneous injection of 50 microm/m2 of G-CSF for 24 h (CI; n = 6) and were followed for 48 h. Circulating leukocyte counts, markers of activation on PMN, and circulating levels of G-CSF, IL-6, and PMN elastase were measured. SI rapidly increased serum G-CSF levels, which peaked at 4 h, whereas CI gradually increased G-CSF levels, which remained at a steady level from 8 to 24 h. SI caused a rapid decrease in PMN counts at 0.5 h followed by sustained increase to peak at 12 h. CI gradually increased PMN counts, which peaked at 24 h, but the peak values were not significantly different between the groups. SI-induced activation of PMN, which was characterized by increased expression of CD11b, decreased expression of L-selectin, and increased F-actin content, led to increases in serum IL-6 and PMN elastase level. Such changes were all attenuated with CI (P < 0.05). We conclude that continuous subcutaneous injection of G-CSF resulted in a marrow response similar to that to a single injection but yielded reduced PMN activation.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

Effect of microvillus deformability on leukocyte adhesion explored using adhesive dynamics simulations

Leukocyte rolling on the endothelium via selectin molecules is an important step in the adhesion cascade, which allows leukocytes in the bloodstream to reach sites of infection. We improve upon Adhesive Dynamics simulations by incorporating deformable microvilli on which adhesion molecules are clustered. As determined in micropipette experiments, microvilli deform like an elastic spring at small forces and a combination of yield and viscous dissipation at high forces. First, we create a modified version of the state diagram for adhesion which includes microvillus deformation, and find four adhesion states-firmly bound; landing; rolling; and no-adhesion. Then, we simulate the effects of receptor clustering on the tips of microvilli, number of adhesion molecules on the cell, and the spring constant of the bonds, within the context of deformable microvilli. We also explore how the microvillus rheology itself controls the dynamics of adhesion. A minimum in rolling velocity occurs at an intermediate value of the microvillus membrane viscosity, remarkably close to the reported physiological value, suggesting that the mechanics of microvilli have evolved ideally for rolling and adhesion of leukocytes. We find that a larger degree of association between the membrane and cytoskeleton leads to slower rolling, and stiffer microvilli result in faster rolling. Decreasing the overall deformability of the microvilli greatly reduces a simulated cell's ability to roll. A comparison to experimental results of in vitro cell rolling agrees with the simulation at low shear rates. Furthermore, simulated rolling trajectories of cells with deformable microvilli display periods of rolling interdispersed with pauses, consistent with that seen in experiments where microvilli were observed to stretch.     

Expression of IgG Fc receptors in myeloid leukemic cell lines. Effect of colony-stimulating factors and cytokines

Three classes of FcR have been defined on human myeloid cells by their reactivity with mAb; FcRI (mAb 32); FcRII (mAb IV3); and FcRIII (mAb 3G8). We have quantitated the expression of each FcR on human myeloid leukemia cells and cell lines (KG-1, HL-60, U937, and K562). Detailed analysis of FcR surface expression is provided for the U937 cell line after exposure to CSF and cytokines. Increased expression of FcRI and FcRII occurred at 72 h in cells exposed to GCT or Mo cell line-conditioned medium as well as to medium from PHA-treated mononuclear cells. The augmentation of FcRII required protein synthesis and was diminished by a neutralizing antibody to granulocyte-macrophage CSF. We also show that fractions containing natural granulocyte CSF or granulocyte-macrophage CSF as well as r-granulocyte and r-granulocyte-macrophage CSF are capable of inducing FcRII on these cells, whereas other cytokines such as IL-1 and IL-2, TNF-alpha, INF-gamma and macrophages CSF failed to do so.     

Effect of passive repetitive isokinetic training on cytokines and hormonal changes

It is well known that muscle strength and power are important factors in exercise. Plyometrics is designed to gain muscle strength and power in a shock method. The passive repetitive isokinetic (PRI) machine is developed for plyometrics. The present study aims to understand the effect of ten-week PRI training in different intensities on human plasma concentration cytokines as well as hormonal changes. Thirty young male subjects were enrolled into the ten-week PRI training program and were divided randomly into traditional, low- and high-intensity PRI training groups. Blood samples were obtained before, during, after and 1-, 2-, 3-, 5- and 7-day (D) post-training. The plasma concentrations of cytokines and hormones were measured by an enzyme-linked immunosorbent assay (ELISA). Elevated plasma IL-2 was found in the subjects in all the training programs. Significant increases of proinflammatory cytokines IL-1beta and TNF-alpha were observed at post 7 D in the high-intensity PRI training (29.5 +/- 4.4 and 515.8 +/- 127.1 pg/ml, respectively). No significance in differences in the plasma concentration of IL-6 was observed in the traditional and low-intensity PRI training. Significant elevation of IL-6 was found at post 5 D in high-intensity PRI training. Higher plasma IL-6 concentration was observed at post 3 and 5 D in high-intensity PRI training compared to low-intensity PRI training (P < 0.05). Significant elevation of plasma IL-15 during (week 6) and after (post 0 D) was observed in low-intensity PRI training. Also, there were differences between low-intensity PRI training and traditional training at post 0, 2, 3, and 5 D. The plasma concentration of cortisol was decreased to the lowest value (118.0 +/- 17.3 ng/ml) at post 0 D in traditional training, then returned to the baseline (220.5 +/- 19.1 ng/ml). In the high-intensity PRI training, but not in the low-intensity PRI training, the cortisol level dropped from 224.9 +/- 25.8 ng/ml at post 0 D down to the 123.2 +/- 22.6 ng/ml at post 1 D. Significant differences were found at post 1 and 5 D between low- and high-intensity PRI training, and post 0, 1, 2, and 3 D between traditional and high-intensity PRI training. Significant increased testosterone was found post 0, 1, 2, and 3 D in traditional training. Higher plasma testosterone was observed during and the recovery period in low-intensity, but not in high-intensity, PRI training. In conclusion, high-intensity PRI training could induce the proinflammatory cytokines, i.e., IL-1beta and TNF-alpha, and decrease plasma cortisol in the recovery period.     

Effect of quercetin on human polymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism

Quercetin inhibited in a concentration-dependent manner the release of beta-glucuronidase from human polymorphonuclear leukocytes stimulated with zymosan-activated serum. ³H-arachidonic acid-prelabelled polymorphonuclear leukocytes released ³H-arachidonic acid upon stimulation with zymosan-activated serum and this was associated with a decrease of radioactivity in the phospholipid fraction as determined by thin layer chromatography. Quercetin inhibited the release of ³H-arachidonic acid. These observations suggest that the zymosan-activated serum stimulus activates phospholipase A₂ and that phospholipase A2 is inhibited by quercetin. Thus, quercetin alters polymorphonuclear leukocyte phospholipid metabolism and responses to stimulation.     

Effect of salmeterol on polymorphonuclear leukocyte (PMNL) chemiluminescence In Vitro

Although beta-agonists remain an important aspect of the treatment of asthma, their role has recently been questioned. Salmeterol has recently been developed as a betaagonist with prolonged bronchodilator action. Using lucigenin-enhanced chemiluminescence, we have shown that salmeterol inhibits this aspect of phagocyte function in vitro in a concentration-dependent manner. However, salmeterol differs from classical beta₂-agonists in that at concentrations between 10^?5 and 10^?3 mol/L, its effects on phagocytes cannot be completely reversed by washing the cells or by propanolol. The effects on phagocytes may not therefore be explicable on the basis of betaadrenergic mechanisms alone.     

Effect of chemotactic factors on hexose transport in polymorphonuclear leucocytes

Transport of the nonmetabolizable glucose analogue, 3-O-methylglucose, was assessed in human polymorphonuclear leucocytes with or without the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). The peptide increased entry of labelled 3-O-methylglucose about 5-fold and the intracellular distribution space about 70%. The half-time of equilibration was 3 s in the treated cells. Similar effects were observed with zymosan-treated serum (containing the chemotactic factor C5a), with arachidonic acid, calcium ionophore A23187 and phorbol myristate acetate. However, the chemotactic protein, thrombin, had no effect, even though binding to high-affinity receptors was demonstrated. Km for zero-trans entry of 3-O-methylglucose was about 1 mM and fMet-Leu-Phe increased Vmax from 5 to about 25 amol.s-1.cell-1. Similar values were obtained from incubations for a few seconds with glucose and 2-deoxyglucose. The rate of 2-deoxyglucose uptake (8 min incubations) was limited by the transport step at substrate concentrations lower than approx. 0.1 mM, whereas the phosphorylation step became rate-limiting at higher concentrations. Thus, 2-deoxyglucose uptake can only be taken as a measure of transport at a tracer concentration. It is concluded that chemotactic factors can, but do not necessarily, increase the maximal transport velocity of hexoses entering the polymorphonuclear leucocyte via the glucose transporter.     

Effect of high-dose sodium selenite therapy on polymorphonuclear leukocyte apoptosis in non-Hodgkin's lymphoma patients

The present study was undertaken to explore the effect of the administration of high doses of sodium selenite on apoptosis in polymorphonuclear leukocytes in patients with non-Hodgkin's lymphoma. Thirty patients with newly diagnosed non-Hodgkin's lymphoma were randomly divided into two groups. Group I was treated with chemotherapy and group II received 0.2 mg/kg/d sodium selenite in addition to chemotherapy. Flow cytometry was used for the monitoring of apoptosis on peripheral blood neutrophils at the time of diagnosis and after treatment in both groups of patients. Sodium selenite administration resulted in a significant reduction in neutrophils apoptosis (82+/-10% vs 32+/-18%, p<0.05) and this was associated with significant reduction in infection rate following chemotherapy (67% vs 20%, p<0.05). Also, significant improvement in cardiac ejection fraction was observed (62+/-4% vs 69+/-5% p<0.05). It is concluded that sodium selenite administration at the dosage chosen acts as a cytoprotective agent, alleviating side effects and immunosuppressive effects of cytotoxic chemotherapeutic agents.     

Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation

The protein kinase C inhibitor C-I reduced superoxide production by human neutrophils in response to phorbol myristate acetate by greater than 50%. In contrast to its effects in oxidative metabolism, 100 microM C-I caused minimal inhibition (5-18%) of lysozyme release in response to phorbol myristate acetate. Enzyme release produced by the formylated oligopeptide FMLP was enhanced by 23-54% in neutrophils pretreated with 100 microM C-I. These findings suggest that protein kinase C activation is not required for phorbol myristate acetate induced enzyme release. Enhancement of FMLP stimulated degranulation by C-I suggests that protein kinase C activation may have inhibitory effects on the release of granule enzymes by human neutrophils.     

Experimental study on filtrability of polymorphonuclear leukocyte suspensions

Filtrability of a suspension of polymorphonuclear leukocytes (PMNs) was examined in a Nuclepore membrane filtration system utilizing a gradually reduced pressure difference with or without an additional negative pressure. The filtration process was continuously recorded using a TV-video system for data analysis. The PMN content in the filtrate was directly measured. The pressure-flow relation was analyzed in terms of the relative resistance of the PMN suspension to that of the suspending medium. The relative resistance of the PMN suspension increased with an increase in the filtered volume until it approached infinity at the level of low pressure difference (2.8 - 0 cmH2O). The remarkable increase in flow resistance was closely associated with the plugging of PMNs in the membrane pores. At high pressure differences (12.8 - 10 cmH2O, 7.8 - 5 cmH2O), the relative resistance increased up to finite values, as the filtered volume increased. The variation in the relative resistance was greatly dependent upon the pressure difference or the flow condition. The amount of filtered cell fraction increased with an increase of additional pressure, indicating that the relative resistance was changed according to the rate of PMN plugging and dislodging in the pores of the membrane.     

In vitro modulation of canine polymorphonuclear leukocyte function by granulocyte-macrophage colony stimulating factor

Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuclear leukocytes (PMN) to endogenous chemoattractants. We found that,in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosanactivated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H₂O₂ production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H₂O₂ production 8–12-fold after 15 minutes. Preincubation of cells with GMCSF (1–100 U/ml) prior to PMA stimulation significantly reduced the H₂O₂ levels induced by PMA. H202 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10–1000 U/ml) was able to elicit 49%–102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was < 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMNin vitro and may actually down-regulate PMN inflammatory responses.Supported by the Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under work unit No. 00082. Views presented in this paper are those of the authors; no endorsement by the Defense Nuclear Agency has been given or should be inferred. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.     

Interaction of dexamethasone and Salmonella enteritidis immune lymphokines on Salmonella enteritidis organ invasion and in vitro polymorphonuclear leukocyte function

Abstract We used an anti-inflammatory dose of dexamethasone (DEX) and Salmonella enteritidis (SE)-immune lymphokines (ILK) followed by oral SE challenge to chicks to determine the effects of these treatments on SE organ invasion and in vitro function of PMNs derived from peripheral blood. Endpoints included percent protection against SE organ invasion, numbers of peripheral blood PMNs, and in vitro PMN adherence, chemotaxis, and SE killing. SE organ invasion was significantly reduced in chicks treated with either ILK alone or DEX + ILK compared to controls. Chicks treated with either DEX alone or DEX + ILK responded with a significant increase in numbers of peripheral blood PMNs as compared to controls, while numbers of PMNs in the peripheral blood from chicks treated with ILK alone were not significantly increased. PMN adherence and percent SE killing by PMNs derived from chicks treated with either ILK alone or DEX + ILK were significantly increased compared to controls. Chemotaxis of PMNs derived from chicks treated with either ILK alone or DEX alone significantly increased 2-fold over control levels. Interestingly, chemotaxis of PMNs derived from chicks that received DEX + ILK was similar to controls. Generally, ILK abated the anti-inflammatory effects of DEX on PMNs in these assays, except for chemotaxis. We interpret these data to suggest that ILK may confer protection to chicks against the early phase of SE organ invasion by inducing an inflammatory response predominated by activated PMNs.     

Effect of cytokines on hypothalamic neuropeptide Y release in vitro

Studies involving altered energy balance states in rodents have demonstrated that hypothalamic neuropeptide Y (NPY) activity is strongly activated in states of negative energy balance, such as periods of dietary restriction or starvation. However, in cancer cachexia, when there is a significant reduction in body weight as a result of appetite loss, leading to loss in fat and lean tissue mass, there is no augmentation in the activity of the hypothalamic NPY system. Therefore, we have examined whether cytokines, interleukin (IL)-1, IL-1beta, IL-6, and tumor-necrosis factor-alpha (TNF-alpha; cachectin), which are elevated in cancer patients, can attenuate NPY release from hypothalamic slices in vitro. None of the cytokines altered either the basal or stimulated NPY release from the hypothalamic slices. However, we were able to measure a significant reduction in potassium-stimulated NPY release (-60%) by using the nonselective voltage-dependent calcium channel blocker NiCl (30 microM) without any effect on basal release, as a positive control. Therefore, we suggest that the failure to activate the hypothalamic NPY system in states of cancer cachexia cannot be attributed to a cytokine-induced reduction in neurotransmitter release.     

The effect of taurine on polymorphonuclear leukocyte functions in endotoxemia

Summary. The aim of the present study was to measure MPO activity in PMN leukocytes after endotoxin administration, and to compare the levels of NO₂ ⁻ competing with taurine for reaction with HOCl. Furthermore we aimed to determine TauCl levels, a product of MPO–H₂O₂–Halide system, and to evaluate anti-inflammatory properties of PMN in endotoxemia. In addition, our second objective was to investigate the effect of taurine, an antioxidant amino acid, on anti-bactericidal and anti-inflammatory functions of PMN after administration of endotoxin together with taurine. All experiments were performed with four groups (control, taurine, endotoxemia, and taurine plus endotoxin) of ten guinea pigs. After endotoxin administration (4 mg/kg), MPO activities increased and taurine levels decreased. Therefore levels of TauCl, NO₂ ^•− increased. We observed the effects of taurine as conflicting. When taurine was administrated alone (300 mg/kg), all of these parameters decreased. Consequently, we suggested that taurine is influential in infected subjects but not on healthy ones as an antioxidative amino acid. In addition, we believe that in vivo effects of taurine may differ from those in vitro depending on its dosage.     

Physico-chemical factors of erythrocyte deformability

The aim of this study is to assess the role of different physico-chemical factors on the deformability measurements by using the initial filtration flow rate method, and to differentiate between the membrane or internal origin of some rigidity changes. The deformability is maximum for the physiological pH value and it decreases sharply for hypotonic and hypertonic buffer. For normal RBC, the deformability is independent of the pO2 level and a small decrease is observed for increasing pCO2 values (with constant pH). A theoretical model of filtration for the "Hemorheometre" will be also developed.     

Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets within a whole blood culture

Pro-inflammatory cytokines are known to affect apoptosis in human peripheral blood cells. Neutrophils, which are an essential component of the immune response and usually undergo apoptosis rapidly, are greatly affected by these cytokines. In this study, the effect of varying concentrations of TNF-alpha, IL-1beta and IL-6 on the apoptotic response of leukocytes and their sub-sets in cultured whole blood were studied over a 48 h culture period. At clinically relevant concentrations, it was found that these pro-inflammatory cytokines reduced the amount of spontaneous apoptosis in neutrophils in culture, but had little effect on the lymphocyte population. Distinct differences in the sensitivity of neutrophils to cytokine-mediated protection against spontaneous apoptosis were apparent when compared to previous studies conducted using purified or enriched neutrophil cultures. IL-1beta, at a dose of 0.01 pg/mL, was observed to significantly inhibit spontaneous neutrophil apoptosis by approximately 90% and 65% at 24 and 48 h of culturing, respectively. This concentration used in whole blood is dramatically lower than that required to elicit similar protection in neutrophil-enriched cell cultures. Higher concentrations of TNF-alpha (1.0 pg/mL) and IL-6 (125 pg/mL) were also found to significantly inhibit neutrophil apoptosis, at levels much lower than previously published using neutrophil-enriched cultures. Furthermore, each cytokine displayed a unique signature with respect to the optimal applied doses required to elicit maximal protection against spontaneous neutrophil apoptosis. These results demonstrate the dramatic differences in cellular responses that exist between neutrophil-enriched cultures and whole blood culture systems, where multiple blood cell types provide a much more complex environment.